RU2706693C2 - Virus vaccine and methods for production thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and concerns a viral vaccine composition for preventing infections caused by Indian strain JEV821564 of Japanese encephalitis virus containing a vaccine antigen, where the vaccine antigen is an inactivated strain JEV821564 of Japanese encephalitis virus, adapted for growth in Vero cells in a disposable bioreactor for collecting live mass of Japanese encephalitis virus. Group of inventions also relates to a method of producing a viral vaccine composition by adapting strain JEV821564 of Japanese encephalitis virus to Vero cells; method for simultaneous purification and inactivation of JEV821564 strain mass collection of Japanese encephalitis virus.

EFFECT: group of inventions provides preventing infections caused by Indian strain JEV821564 of Japanese encephalitis virus.

12 cl, 4 tbl, 4 ex

Description

FIELD OF THE INVENTION

The present invention relates to the field of viral vaccine compositions. In particular, the present invention relates to a vaccine composition against Japanese encephalitis and to processes or methods for producing a vaccine composition against Japanese encephalitis infections. The present invention relates to processes for the preparation and purification of viral mass and methods for inactivating said viral mass of Japanese encephalitis virus.

BACKGROUND OF THE INVENTION

Virus Japanese encephalitis (JEV) is a mosquito-borne infection, a leading cause of viral encephalitis throughout Asia, and it spreads beyond its continental borders. JEV belongs to the family of flaviviruses and the genus of flaviviruses. It is a small envelope virus (50 nm) containing a negative sense RNA genome in the form of a single chain 10.9 kb in size. Virion RNA encodes 11 proteins, including four structural and seven non-structural proteins. Among the 4 structural proteins, the surface coat protein (E-protein) serves as a protein that binds cell receptors and a fusion protein for virus attachment and entry into the host. Thus, antibodies against E-protein neutralize the virus and play an important role in protecting against it. Surface structural E-protein is an important component of the vaccine, so it is very important that the stabilization of this protein molecule is a suitable stabilizer for the effectiveness of the vaccine.

Due to the wide distribution area of vaccines and the variety of ambient temperatures, there is a need to improve the bioprocess of the production of the viral mass of the Japanese encephalitis virus for the production of a microorganism for the production and further use of vaccines. The bioprocess in the case of Japanese encephalitis involves either growing in rotating bottles or cell factories. The production of commercial quantities of vaccine against Japanese encephalitis by culturing cells in rotating bottles or using cell factories requires longer periods of time with relatively lower yields, as well as occupying a large amount of space, which makes it difficult to control and regulate experimental parameters on a regular and constant basis in each cell the factory. The production of the viral mass is thus inherently devoid of uniformity in the process. The present invention overcomes these difficulties through new bioprocess technology, where the time required to obtain high quantities of industrial viral mass of Japanese encephalitis virus is significantly reduced. Commercial production of the vaccine mass is provided over a significantly shorter period of time compared with previous cultivation methods. Vero cells are first grown in a disposable bioreactor, followed by infection with JEV, where multiple virus harvests are performed as a new feature of the present invention. After production of the virus, methods for simultaneous inactivation and stabilization are followed, in particular those applicable to the Japanese encephalitis vaccine antigen. Stabilization of the Japanese encephalitis vaccine typically requires maintaining the vaccine vials or mass at predetermined temperature ranges of 2 ° -8 ° C to stabilize over longer periods of time. This requires additional infrastructure for transporting and storing the vaccine, thereby increasing the cost of vaccines. Alternative methods of stabilization are required to overcome some of the most practical limitations for Japanese encephalitis virus. The vaccine thus obtained from the viral mass in accordance with the methods described in the framework of the present invention does not require any special refrigeration equipment mentioned above, and is stable under ambient temperature conditions, i.e. at room temperature (25 ° C). The stability achieved by the present invention is comparable to that observed for Japanese encephalitis vaccines stored at refrigerated temperatures (2 ^° -8 ^° C). The effectiveness and immunogenicity of the viral mass and vaccine to ensure sufficient immunization of individuals are also preserved.

OBJECTS OF THE INVENTION

One of the objectives of the present invention is to obtain a stable inactivated vaccine composition, which is able to provide the prevention and treatment of Japanese encephalitis infections caused by the Indian Kolar strain of Japanese encephalitis virus.

Another objective of the invention is to develop a method of adaptation of the Kolar strain of Japanese encephalitis virus in Vero cells.

Another objective of the invention is the development of a suitable method for the commercial production of purified inactivated viral mass of the Japanese encephalitis virus at specified experimental parameters, and methods that provide multiple collection of the viral mass of the Japanese encephalitis virus with significantly higher yields using a disposable bioreactor.

One or more objectives of the invention are the development of new cleaning methods using a column with a specialized matrix for the subsequent processing of the viral mass of Japanese encephalitis virus on a commercial scale.

Another objective of the invention is to develop a method for the simultaneous inactivation and stabilization of the purified viral mass of the Japanese encephalitis virus.

SUMMARY OF THE INVENTION

According to one embodiment of the invention, various methods for culturing Vero cells in a disposable bioreactor are described. Satisfactory Vero cell growth levels under various experimental parameters involving the proper composition of the medium and the need to replace the medium are optimized to obtain satisfactory cell numbers over the desired amount of time. Vero cells are infected with the live Japanese encephalitis virus of the Indian Kolar strain, and thus, in this embodiment, a method for adapting Japanese encephalitis virus to growth in Vero cells in a disposable bioreactor is described.

According to another embodiment of the invention, the adaptation of the Kolar strain of Japanese encephalitis virus in Vero cells is scaled from a single-use bioreactor to the following sizes of single-use bioreactors to ensure yield of a purified batch-sized JEV mass. Disposable bioreactors are designated as follows: from DB-1 to DB-100 to DB-500, depending on cell capacity.

In a further embodiment, alternative methods for the subsequent purification of JEV live weight are described using various new chromatographic methods that provide high amounts of purified JEV mass.

In one additional embodiment of the invention, new methods for simultaneously inactivating and stabilizing a purified JEV mass are described.

The invention also relates to experimental data on the stability of the purified inactivated mass of JEV at 25 ° C-37 ° C, which is ready for the vaccine composition.

In a further embodiment, the invention provides a Japanese encephalitis vaccine composition comprising a vaccine antigen, wherein the antigen is a Kolar strain of Japanese encephalitis virus.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

Initial cell culture: initial number of cells required for culture on a culture vial.

DMEM: Dulbecco Modified Needle Medium

EMEM: Minimum Support Environment Needle

NBCS: Newborn Calf Serum

In the framework of the present invention, a new Indian strain of Japanese encephalitis virus JEV821564-XY, which is a Kolar strain of Japanese encephalitis virus, is described. This Japanese encephalitis virus Kolar strain JEV821564 (hereinafter referred to as the "Kolar strain") was transported to Bharat Biotech International Limited according to the Material Transfer Agreement from the National Institute of Virology, Pune. There is currently no vaccine composition against the Indian strain (Kolar) of Japanese encephalitis virus. Moreover, this particular strain of Japanese encephalitis virus is grown by a method in which the strain is adapted in Vero cells for cultivation. Viral culture is usually grown in liquid media. The cell culture in Vero cells for any biological product includes a very high number of rotating vials or cell factories. Cultivating the virus in cell factories is time consuming. Cell factories occupy large spaces, and it is also difficult to individually monitor all experimental parameters during the cultivation of the virus in cell factories. The production of Japanese encephalitis virus using conventional cell factories is minimal.

Thus, the present invention relates to a new method for the production of this Kolar strain of Japanese encephalitis virus in a disposable bioreactor. Japanese encephalitis virus is adapted for growing in Vero cells, and this particular strain of the virus is adapted for propagation in Vero cells provided with specific media. The same Vero cells are able to provide multiple collection of viral mass in a single infection. This particular Japanese encephalitis virus (JEV) multiple collection method described in the framework of the present invention is unique and individual, although an increase in the amount of JE virus in Vero cells is already known. Moreover, achieving the desired level of cell growth (Vero cells) in a new device (other than rotating bottles and cell factories), such as the device used in the framework of the present invention, which is a disposable bioreactor, and scaling from disposable bioreactors of smaller capacity to size Disposable bioreactors for batches with higher capacities have not been provided previously. A commonly used method for increasing the amount of Japanese encephalitis virus in Vero cells is usually grown in cell factories or rotating bottles in which Vero cells are used to form a layer only once. The Vero cell line is used by Japanese encephalitis virus infection, and the cell line is depleted after only one virus inoculum infection. However, in the framework of the present invention, Vero cells are grown using such a unique and new method and the experimental parameters are set in such a way that a single inoculation of the Japanese encephalitis virus inoculum can produce at least three to five times the viral mass from the same cell line culture Vero using a disposable bioreactor. At the same time, this reduces the number of infections, as well as the amount of space and, moreover, the number of cell factories required to infect and grow the same amount of viral cell culture. In addition, multiple harvests from cell culture also automatically increase the yield, both in terms of quantity and in terms of the quality of the collection of the virus, to very high quantities compared to the processes involved in providing and achieving similar yields with cell factories.

Moreover, the present invention is directed to the stabilization of the purified JEV mass both during inactivation and during storage of the viral mass. The content of each stabilizer is in the range from 0.5 to 1% wt./about. Chemical stabilizers that are added to the viral antigen are D-sorbitol and glycine. A viral antigen with a suitable stabilizer is stored in a sufficient physiologically acceptable phosphate-saline buffer to maintain a pH of from 7.0 to about 7.4.

Example 1: Cell culture and adaptation of the Kolar strain of JE virus to Vero cells

The initial plating of Vero cells is carried out in a T-175 vial containing Vero cells. Vero cells are cultured in T-175 vials and grown in a suitable medium. The grown cells obtained from 1 T-175 vial are transferred to 5 T-175 vials after 4-5 days. Then they are transferred to 1 cell factory 10 (CF10) again after 4-5 days, and then again transferred to 5 cell factories 40 (CF-40). CF-40 is currently used for commercial mass production of JEV. One batch of JEV pulp involving at least 30 CF-40 is produced within one month to produce one commercial batch of JEV pulp. The present invention relates to a specific method for increasing the amount of said Kolar JE strain in Vero cells, suitable for commercial scaling of mass production, and it is capable of producing JEV mass up to about 140 CF-40 in one time period, i.e. in about one month. This leads to higher virus yields, since the total amount of extracted virus mass is very high compared to 30 CF40. Such a high mass yield of JEV over the same period of time is a great advantage for a commercial increase in productivity. Moreover, it is practically impossible to monitor and control approximately 200 CF40 at a time for a period of time, i.e. for about one month. In accordance with modern practice, to maintain a batch size of 30 CF-40, the minimum requirement for a cultivation room is a room type incubator, which can accommodate at least 3-4 people for operation and experimentation. In a room type incubator, a constant set temperature of 37 ° C should be maintained during batch cultivation. Thus, it can be stated that the need for a room type incubator to work continuously for a period of 5-6 months, which can provide the output of 200 cell factories 40 (200 CF40), will definitely increase the cost of production. Alternatively, operating up to about 150 CF-40 at a time for one month will require a huge and extra-large room-type incubator, maintained constantly at 37 ° C, to provide enough space for at least 20 people to operate and monitor experimental parameters. The construction of such a room is not at all economical. Such a huge room-type incubator will involve additional and very large investments, which will make the vaccine inadmissible for those who are most in need of a vaccine against JE. All experimental parameters will vary and the final results of the experiment cannot be achieved at the same time to isolate the mass of the Japanese encephalitis virus, i.e. for producing a vaccine. Cell cultivation in 150-200 CF40 will take more than about 6 months for an equivalent level of JEV mass production. Important experimental parameters, such as temperature, pH, dissolved oxygen, mixing, and medium flow, cannot be controlled simultaneously for 150-200 CF40 for one month. Moreover, monitoring all of these experimental parameters of 30 CF40 in a single batch of JEV mass is also very complex and requires highly developed skills and human resources and labor to ensure successful production of the required amount of JEV mass. Unlike the CF40 situation, the present invention describes a new method for producing JEV pulp in a disposable bioreactor, but not in cell factories (CF40), where all the technical characteristics of the disposable bioreactor can be controlled with the required accuracy and precision that are required for cell culture and viral culture that cannot be achieved in the case of cell factories. The present invention replaces the use of CF40 in the production of JEV mass with such a suitable disposable bioreactor. This disposable bioreactor provides the necessary channels, probes and sensors, through which it is possible to regulate the process of cell culture and virus cultivation automatically. Thus, the control of various experimental parameters, such as temperature, pH, dissolved oxygen, shaking and feeding of the medium, is easy to carry out in this disposable bioreactor with the required accuracy and accuracy. In addition, adaptation of the Kolar strain of JE virus in Vero cells in a bioreactor is capable of producing JEV mass to an amount of at least 40 CF40 to 200 CF40 while producing one batch at the same time as 30 CF40 in one batch. The yields achieved are generally higher both in the case of equivalent corresponding output and output of 30 CF40, and obviously they are very high in the case of corresponding output, and have products that would be achieved by up to 200 CF40 within 5- 6 month. Also, if bioreactors are used instead of CF40, the level of losses is significantly minimized by a substantial amount. It is obvious that in the case of production involving approximately 200 cell factories, a loss of batch production must exist for a period of 5-6 months. This possibility is completely eliminated using a bioreactor to produce JEV mass according to the methods described in the framework of the present invention. In the experiments presented below, it is shown that the present invention, which consists in adapting a specific Kolar strain of JE virus in Vero cells grown in a bioreactor, increases the yield of the virus when comparing the production level with both 30 CF40 and / or 200 CF40. In addition to the above-mentioned advantages of producing JE pulp in a disposable bioreactor, one additional advantage is that the risk of contamination is minimized to a large extent in the case of disposable bioreactors, as described in the framework of the present invention. Since all experimental parameters are controlled by electronic instructions, manual operation is minimal for mass production of JE in a disposable bioreactor. Thus, unlike cases of batch production in cell factories where there is a higher likelihood and possibility of batch contamination, this is not applicable for mass production of JE in disposable bioreactors of various sizes, designated as DB, DB-100 and DB-500, as described below in the following paragraphs. One further advantage of a disposable bioreactor is that it can produce cells continuously, thereby providing a continuous output, while in the case of cell factories it is not possible to maintain continuous output to a similar extent with the output of disposable bioreactors due to manual control to regulate experimental parameters.

Typically, the total available surface area for cell growth in 30 CF40 is 763200 cm ² , which may correspond to a capacity of ~ 75000-90000 x10 ⁶ cells, while the disposable bioreactor used in the framework of the present invention have a total surface area available for cell growth, ranging from 26,000 cm ² to 5,000,000 cm ² , which supports the growth of the required number of cells and the continued availability of Vero cells as a substrate for the virus, so that it can infect an increasing number of Vero cells so that it reaches the required quantities for increased mass production of JE in the bioreactor. Total cell capacity ranges from approximately 7,800 × 10 ⁶ to 1,500,000 × 10 ⁶ cells. An increase in the number of available cells means an increase in the available substrate for the virus to infect an increasing number of cells, and thus a higher level of mass production of JE can be achieved.

Table 1.1 Comparison of cell factories and disposable bioreactor (s) MODEL OF CULTURE Available Vero Cell Growth Area Device Amount of time required to grow one batch The total size of the cell capacity for growing = the number of cells available for infection with Vero JE virus Bottle T175 175 cm ² ---- ---- (20-30 × 10 ⁶ ) 1 CF10 = 36 T-175 Vials 36 × 175 = 6300 cm ² ---- ---- (600-900 × 10 ⁶ ) 1 CF40 = 4 CF10 4 × 6300 = 25200 cm ² Disposable Bioreactor (DB) 1 CF40: (2500-3000 × 10 ⁶ )
DB: (7800 × 10 ⁶ ) 30 CF40 30 × 25200 = 756000 cm ² Room type incubator containing cell factories for one commercial batch 40 days 30 CF40:
(75000-90000 × 10 ⁶ ) 40 CF40 40 × 25200 = 1008000 cm ² Disposable Bioreactor-100
(DB-100) About 45 days in cell factories.
Approximately 38 days in the DB-100. 40 CF40:
(100000-120000 × 10 ⁶ )
DB100: (300000 × 10 ⁶ ) 200 CF40 200 × 25200 5040000 cm ² Disposable Bioreactor-500
(DB-500) About 280 days in cell factories.
Approximately 38 days in DB-500 200 CF40:
(500000-600000 × 10 ⁶ )
DB500: (1500000 × 10 ⁶ )

Cell culture is a widely used method in biotechnological processes. However, although a given cell is capable of growing in more than one given medium, its characteristics may change when the medium is replaced. It is also important to note that with respect to the cell line, the time and duration of cell growth also vary. Cell growth depends on several important factors, such as environment, growth factors, glucose concentration, oxygen availability, and the size of the initial inoculum and the multiplicity of infection (MOI). Moreover, cell growth is also subject to irregular pH changes, which requires constant monitoring during various phases of cell culture. Thus, it has already been established that cell growth is specific in relation to the purpose, and environmental conditions, and physico-chemical factors. The cultivation of the Vero cell line in the aforementioned bioreactor instead of cell factories, in contrast to previous practice, is a separate problem that was solved in the framework of the present invention. In addition, growing a Vero cell line in a bioreactor may require frequent medium changes at specific and suitable time intervals for optimal cell growth. Replacing the environment also requires specific calculated economic measures to maintain maximum cell growth. Controlling temperature fluctuations in a bioreactor is also an important issue to maintain good cell growth. At the same time, a very high level of cell growth can lead to a low yield, while in the case of very low cell growth below the desired levels, the result of experiments is also a failure. Thus, it is desirable to amplify and tune all of the above parameters in a new environment (i.e., a disposable bioreactor) to achieve the required number of healthy Vero cells that will provide the best JE mass yields. Thus, a series of experiments were performed to grow Vero cells in a microcarrier-based cell culture in this suitable disposable bioreactor, and cell growth in the appropriate medium was observed and tabulated according to the corresponding number of days. Experiments were noted in which more than 100,000 cells / cm ² and the corresponding day from the initial seeding were successfully achieved. The results were further used to infect Japanese encephalitis virus in corresponding experiments with a multiplicity of infection (MOI) of 0.005.

Adaptation of a particular strain of the virus to a particular cellular substrate is also a problem. Although adaptation of JE virus on Vero cells is known, it is nevertheless always strain specific. Each individual strain of the virus involves specific conditions for the growth and infection of Vero cells in a given way, which ensures high growth and rapid increase in the number of viruses. The invention described herein relates to the adaptation of the Kolar strain of Japanese encephalitis virus to Vero cells. Characterization of a laboratory-adapted strain of the JE virus may exhibit characteristics that differ from the characteristics of the previously isolated strains in terms of rapid growth of Vero cells, virus titer and their final efficiency. Virus characteristics and genetic stability can determine the yield and necessary processes required for commercial production of JE mass. The difficulties associated with the different strains of the virus cannot be directly correlated with earlier adapted processes. Thus, the present invention describes the adaptation of a new strain of Japanese encephalitis virus, which is a Kolar strain of JE virus, isolated and endemic to the Indian subcontinent, to Vero cells in a specialized disposable bioreactor, as mentioned in the examples described above, which is capable of high yield JE masses with high efficacy sufficient to induce a protective immune response in these individuals against Japanese encephalitis virus infections. In the examples presented in the framework of the present invention, a unique method is described for adapting a new strain of Kolar Japanese encephalitis virus to Vero cells, and its multiple collection from the original cell line. The experiments detailing the repeated collection of viruses are described in the experiments mentioned below and are tabulated as follows (table 1.2) for a one-time bioreactor (DB) and further scaling in DB-100 (experiments I-II) and DB-500. Although cell cultivation is carried out in cell factories, cell culture is gradually, sequentially and chronologically scaled from T-175 vials to cell factories in this order: from 1 T-175 to 5 T-175 to 1 CF10 to 5 CF10 to 5CF40 to 30CF40. Transferring live Vero cell lines from one platform to the next platform requires trypsinization. Trypsinization is the process by which attached cells are forced to detach from the monolayer so that they can be used at the next level, which is called cell expansion. Such trypsinization is a key process, and it requires skills to carry it out, and trypsinization at each specific intermediate stage is time-consuming. Initial experiments were conducted with the initial seeding of cells, consisting of 26,000 cells / cm ² in a disposable bioreactor original volume is referred to as the DB, showed that, despite the use of DB100 and DB500, trypsinization still need some time before the initial seeding of cells in disposable bioreactor. Thus, it is necessary to minimize the seeding of cells in a single-use bioreactor, since it is directly proportional to large-scale seeding in the bioreactor. For example, to optimize inoculation of 26,000 cells / cm ² for a single-use bioreactor (DB) of the original size, then inoculation in a DB-100 would require 10 CF-40 grown cells (26,000 × 10 ⁶ cells), where 50 would be required for a DB-500 CF-40 cells (130,000 × 10 ⁶ cells). It is highly difficult to manipulate these many trypsinization CFs to collect cells from them. With a modified or minimized culture of 3000 cells / cm ^{2, the} DB-100 requires 1 CF-40 grown cells (3000 × 10 ⁶ cells), where the DB-500 will require 6 CF-40 cells (15000 × 10 ⁶ cells).

Experiments were performed with a disposable bioreactor with an initial cell culture of 3000 cells / cm ² with EMEM, glucose 1 g / L. For multiple cell growth, the medium was replaced and maintained at an approximate glucose concentration of approximately 1 g / L. The number of cells multiplied gradually, but the growth rate was slower and time-consuming, slowing down the infection with the virus, which further led to a low virus collection after the first and second harvests. Thus, subsequent experiments were performed to achieve the goal of minimizing initial cell seeding to approximately 3000 cells / cm ² in a single-use bioreactor, so that significant Vero cell growth was achieved, along with appropriate provision of medium such as DMEM and high glucose concentrations ( 4.5 g / l). Achieved a higher growth rate compared to previous experiments with optimized environments, also not requiring replacement of the medium.

(Experiments III-IV) are presented in the table below:

Table 1.2 Scaling from a single bioreactor (DB) to DB-100 and DB-500 and the magnitude of multiple virus collection. All virus collection data are presented in plaque forming units. Experiment I in the DB-100 Experiment II in DB-100 Experiment III in the DB-500 Experiment IV in the DB-500 Cell culture 3000 cells / cm ² 3000 cells / cm ² 3000 cells / cm ² 3000 cells / cm ² Vh1 10 ^6.88 10 ^6.75 10 ^7.22 10 ^7.41 Vh2 10 ^7.02 10 ^7.16 10 ^6.92 10 ^7.11 Vh3 10 ^6.49 10 ^6-53 10 ^6.41 10 ^6.47 Vh4 10 ^5.84 10 ^6.01 10 ^5.84 10 ^5.78 Used environment EMEM, 1.5 g / l glucose DMEM, 4.5 g / l glucose EMEM, 1.5 g / l glucose DMEM, 4.5 g / l glucose JE virus infection on 5 days 5 days 7 days 7 days MOI 0.005 0.005 0.005 0.005 The number of cells / cm ² Media Replacements Without changing the medium, maintaining glucose at approximately 1 g / l Without changing the medium, maintaining glucose at approximately 1 g / l Without changing the medium, maintaining glucose at approximately 4.5 g / l Without changing the medium, maintaining glucose at approximately 4.5 g / l Table 1.3 The operating parameters of the disposable bioreactor for experiments conducted according to the above table 1.2 S. No Description Range one Dissolved oxygen in ppm 50-70 2 pH 7.2-7.6 3 Temperature in ° C 35.5-37.5 4 Shaking in rpm 260-280 5 Inlet flow rate in ml / min 80-150 6 Outflow rate in ml / min 84-156

Tasks and results of each experiment

Experiment I - Task: Scaling from a disposable bioreactor to a disposable bioreactor 100 (DB-100) of a commercial batch size.

This experiment was carried out with a disposable bioreactor 100 (DB100) with an initial seeding of cells of 3000 cells / cm ² with EMEM, glucose 1.5 g / L. The medium was not replaced for cell growth, and the glucose concentration was maintained at about 1.0 g / L. The medium also contained 5% NBCS (moregate). A gradual increase in the number of cells occurred. On day 5, it reached a maximum of 100,000 cells / cm ² . The Kolar strain of JE virus was obtained for infection of Vero cells on day 5 with an MOI (multiplicity of infection) of 0.005. Virus 1 (VH1) was harvested using serum-free medium 4 days after infection and VH2, VH3 and VH4 were collected at 3 days intervals, respectively, starting from the day of infection. The collected viruses were purified by microfiltration or using depth filters. The purified collected viruses were concentrated using a 300-kDa flow filtration cassette along the stream, and the concentrated collected virus was further purified on a column.

Results: Individual dash values of the collected virus are mentioned in the table above. The desired increase in virus titer has been achieved. No media change required. VH1 showed a titer value of 10 ^6.88 bp and VH2 showed a titer value of 10 ^7.02 bp VH3 and VH4 titers showed 10 ^6.49 bp and 10 ^5.84 bp, respectively.

Experiment II - Task: Scaling from a disposable bioreactor to a disposable bioreactor 100 commercial batch sizes

This experiment was performed with a disposable bioreactor 100 (DB100) with an initial seeding of cells of 3000 cells / cm ² with DMEM, glucose 4.5 g / L. The medium was not replaced for cell growth, and the glucose concentration was maintained at about 1.0 g / L. The medium also contained 5% NBCS (moregate). A gradual increase in the number of cells occurred. On day 5, it reached a maximum of 100,000 cells / cm ² . Cells were infected on day 5 with an MOI of 0.005. Virus 1 (VH1) was harvested using serum-free medium 4 days after infection and VH2, VH3 and VH4 were collected at 3 days intervals, respectively, starting from the day of infection. The collected viruses were purified by microfiltration or using depth filters. The purified collected viruses were concentrated using a 300-kDa flow filtration cassette along the stream, and the concentrated collected virus was further purified on a column.

Results: Individual dash values of the collected virus are mentioned in the table above. The desired increase in virus titer has been achieved. No media change required. VH1 showed a titer value of 10 ^6.75 bp and VH2 showed a titer value of 10 ^7.16 bp VH3 and VH4 titers showed 10 ⁷ b.p.

Experiment III - Task: Scaling from a disposable bioreactor 100 (DB100) to a commercial bioreactor 500 (DB500) of a commercial batch size.

This experiment was carried out with a disposable bioreactor 100 (DB100) with an initial seeding of cells of 3000 cells / cm ² with EMEM, glucose 1.5 g / L. The medium was not replaced for cell growth, and the glucose concentration was maintained at about 1.0 g / L. The medium also contained 5% NBCS (moregate). A gradual increase in the number of cells occurred. Cells were infected on day 5 with an MOI of 0.005. Virus 1 (VH1) was harvested using serum-free medium 4 days after infection and VH2, VH3 and VH4 were collected at 3 days intervals, respectively, starting from the day of infection. The collected viruses were purified by microfiltration or using depth filters. The purified collected viruses were concentrated using a 300-kDa flow filtration cassette along the stream, and the concentrated collected virus was further purified on a column.

Results: Individual dash values of the collected virus are mentioned in the table above. The desired increase in virus titer has been achieved. No media change required. VH1 showed a titer value of 10 ^7.22 b.p.u. and VH2 showed a titer value of 10 ^6.92 bp VH3 and VH4 titers showed less than 10 ⁷ bp

Experiment IV - Task: Scaling from a disposable bioreactor 100 (DB100) to a commercial bioreactor 500 (DB500) of a commercial batch size.

This experiment was performed with a disposable bioreactor 100 (DB100) with an initial seeding of cells of 3000 cells / cm ² with DMEM, glucose 4.5 g / L. The medium was not replaced for cell growth, and the glucose concentration was maintained at about 1.0 g / L. The medium also contained 5% NBCS (moregate). A gradual increase in the number of cells occurred. Cells were infected with the Kolar strain JE virus on day 7 with an MOI of 0.005. Virus 1 (VH1) was harvested using serum-free medium 4 days after infection and VH2, VH3 and VH4 were collected at 3 days intervals, respectively, starting from the day of infection. The collected viruses were purified by microfiltration or using depth filters. The purified collected viruses were concentrated using a 300-kDa flow filtration cassette along the stream, and the concentrated collected virus was further purified on a column.

Results: Individual dash values of the collected virus are mentioned in the table above. The desired increase in virus titer has been achieved. No media change required. VH1 demonstrated a titer value of 10 ^7.41 bp and VH2 showed a titer value of 10 ^7.11 bp VH3 and VH4 titers showed 10 ^6.47 bp and 10 ^5.78 bp, respectively.

Example 2: Purification of JE mass using a Celufine sulfate ester matrix and inactivation of JE mass

Purification of the JE mass produced in a disposable bioreactor was carried out using a Celufine sulfate affinity chromatography column. Celufine sulfate column purification involves a chromatographic method in which said celufine sulfate is a column matrix made from a cellulosic matrix substrate in the presence of an ester activated sulfate group. The total collection volume is first concentrated using a 300-kDa membrane from 300 liters to 50 liters to give a retentate containing live JEV. The filtrate is discarded. The retentate contains the JE virus. The retentate is purified on a column using the indicated Celufine sulfate matrix. A concentrated volume of the collected virus of 50 liters is passed through a Celufine sulfate column and the fraction passing through the column is discarded because it contains undesirable protein and other genetic material. The virus that binds to the column is eluted using an elution buffer containing high amounts of salts. The eluate is quantified using measured absorbance values giving a sharp peak, which indicates the presence of purified live Japanese encephalitis virus. The virus collected by elution is diafiltered to remove salts, certain unwanted proteins and other genetic material by replacing the buffer against phosphate-saline buffer repeatedly for at least 5-6 times. The retentate is collected and a 0.45 μm PVDF (polyvinylidene fluoride) membrane filter is passed. Then the live weight of JE is inactivated with formalin in a ratio of 1: 1500 vol./about. up to 1: 2500 rpm for 7 days at 22 ° C together with a mixture of stabilizers containing 0.1-1% sorbitol and 0.1-1% glycine, added only during inactivation. Stabilizers are added to the live JE virus at a specific concentration of 0.5% w / v. glycine and 1% wt./about. sorbitol together with an inactivating agent, which is either formalin or BPL. Stabilizers are added to prevent degradation of the virus only during the incubation period for inactivation of 7 days, as mentioned above, due to the addition of an inactivating agent formalin or beta-propiolactone (BPL). After inactivation, the JE mass is then diafiltered by replacing the buffer at least 5-6 times against phosphate-saline buffer to remove formalin from the purified inactivated JE mass. The diafiltered mass of JE was sterilized by filtration using a 0.22 μm PVDF (polyvinylidene fluoride) membrane filter. The inactivated JE mass is stored for stability studies at 5 ° C ± 3 ° C for at least 24 months to 36 months. The stages of purification and inactivation of the mass of the virus of Japanese encephalitis (Kolar strain) in accordance with one of the methods described in the framework of the invention can be summarized below:

(a) purification of the virus collection using a 0.45 μm membrane to remove cell debris from the virus collection;

(b) concentrating the collection volume at least 4 times using a 300-kDa polyethersulfone membrane cartridge (PESU) to obtain a concentrated retentate;

(c) purification on a column of concentrated collected retentate of step (b) using a Celufine sulfate matrix column column, wherein the matrix consists of a matrix with a cellulose support in the presence of an activated group sulfate ester to collect the eluate containing the desired virus, a high amount salts of cellular proteins and nucleic acid material;

(d) diafiltration and buffer replacement using a 300-kDa PESU membrane cassette against the phosphate-salt buffer of the eluate of step (c) for at least 5-6 times to collect retentate to remove an undesirable high amount of salts of cell proteins and nucleic material acids from a virus of interest to collect a purified concentrated virus mass;

(e) Filtering the purified concentrated viral mass of step (d) through a 0.45 μm membrane of PVDF (polyvinylidene fluoride) to remove any impurities in the process;

(f) the simultaneous stabilization and inactivation of the purified virus of stage (e) formalin in a ratio of from 1: 1500 vol./about. up to 1: 2500 rpm (i.e., 1 volume of formalin is added to 2500 volumes of live JE) together with stabilizers, where these stabilizers consist of glycine and sorbitol, for a period of 7 days at 22 ° C, or with beta-propiolactone in a ratio of 1: 1500 r ./about. up to 1: 2500 rpm (i.e., 1 volume of formalin is added to 2500 volumes of live JE) together with stabilizers, where said stabilizers consist of glycine and sorbitol, for a period of 7 days at 5 ° C ± 3 ° C;

(g) diafiltration and replacement of the inactivated purified virus mass buffer of step (f) using a 300-kDa cassette with a PESU membrane against phosphate buffered saline at least 5-6 times to remove only formalin if formalin inactivated;

(h) Filtration through a 0.22-micron membrane from PVDF (polyvinylidene fluoride) of the purified inactivated viral mass of step (g) to obtain a final and stabilized JEV mass at 5 ° C ± 3 ° C for at least 24 months to 36 months free of any impurities, suitable for the preparation of a vaccine against Japanese encephalitis.

Celufine sulfate matrix cleaning results are provided for products with a batch size of 30 CF40 by repeated collection of the virus. The total viral mass obtained for one batch of 30 CF40 using Celufine sulfate in one month is listed below. Thus, the number of doses of JE vaccine that can be available with an NMP of (at least) 5 μg of inactivated JE per dose also increases significantly from at least 60,000 doses in the case of 30 CF40 to at least 1 hundred thousand doses to 3 hundreds thousand doses in a one-time bioreactor 100 (DB-100) and a one-time bioreactor 500 (DB-500), respectively.

Table 2.1 The purified inactivated mass of JEV and the number of doses for 1 month using 30 CF40 with purification on a Celufine sulfate matrix. Collection Details Collection Volume (L) Caption in bp Final mass The mass concentration of JE in μg / ml Total mass JE in mg Vh1 300 10 ^7.45 3.8 L 67.65 257.1 Vh2 300 10 ^7.39 1.8 L 60.23 108,42 Vh3 300 10 ^6.17 1.8 L 36.17 65.12 Vh4 300 10 ^4.35 1,0 l twenty twenty Total 450.64

The total viral mass obtained for one batch of DB100 in one month is listed below. Thus, the number of doses of JE vaccine that can be accessed using 7 μg of inactivated JE per dose is also calculated and presented below:

Table 2.2 The purified inactivated mass of JEV and the number of doses for 1 month using DB-100 with purification on a Celufine sulfate matrix. Collection Details Collection Volume (L) Caption in bp Final mass Protein concentration in μg / ml Total mass JE in mg Vh1 300 10 ^6.54 3.4 l 191.4 650.76 Vh2 300 10 ^6.03 2.0 l 35.9 71.8 Vh3 300 10 ^5.49 1.2 l 28.1 33.7 Total 756.26

The total viral mass obtained for one batch of DB-500 in one month is listed below. Thus, the number of doses of JE vaccine that can be accessed using 7 μg of inactivated JE per dose is also calculated and presented below:

Table 2.3 The purified inactivated mass of JEV and the number of doses for 1 month using DB-500 with purification on a Celufine sulfate matrix. Collection Details Collection Volume (L) Caption in bp Final mass Protein concentration, μg / ml Total mass JE in mg Vh1 500 10 ^7.41 8.0 237.6 1900 Vh2 500 10 ^6.8 6 68.3 409.8 Vh3 500 10 ^6.2 3,7 48,2 178.3 Total 2488.1

Thus, it can be seen that the total mass of JE produced in one commercial batch in the DB-100 disposable bioreactor is equivalent to the amount of JE that would be produced in at least 48 CF40 over the same time period or even less (38 days ) Similarly, in the case of a commercial batch using DB-500, the amount of JE mass produced is equivalent to the amount of JE mass that would be produced in at least 147 CF40 over the same time period (39 days). This provides a significant technical achievement, achieved by conducting commercial production of Japanese encephalitis virus in new disposable bioreactors of sizes DB-100 and DB-500.

Example 3: Purification of JE virus using an agarose matrix column and inactivation of JE virus

Purification of JE virus using a Capto Core-700 agarose column is another new aspect of the present invention. The number of doses that can be produced in a single batch of sizes DB-100 and DB-500 are even higher compared to purification on a Celufine sulfate matrix. The Capto core 700 column consists of a highly crosslinked agarose matrix with a functionally active octylamine ligand, both responsible for hydrophobic interactions and positively charged for strong interaction with most impurities over a wide range of pH and salt concentrations. In this method of purifying the JE virus using the Capto Core-700 matrix, the advantage is that concentration of virus collection is not required. The principle involving the Capto Core matrix is size exclusion along with affinity chromatographic methods, both of which act simultaneously. The columns are cleaned by passing the entire collection volume through the Capto Core matrix. Proteins having a molecular weight below 700 kDa and other genetic material present in the collection bind to the Capto Core matrix. Collect the past fraction containing the live JE virus because the viruses do not bind to the Capto Core matrix. This is followed by concentration through a 300-kDa cassette with a polyethersulfone membrane (PESU). The filtrate is discarded, and the retentate contains live JE virus at this stage. After that, the concentrated live JE virus present in the retentate is chemically inactivated by treatment with the inactivating agent formalin or beta-propiolactone (BPL) in a ratio of 1: 1500 v / v. up to 1: 2500 rpm (i.e., 1 volume of formalin is added to 2500 volumes of live JE) over a period of 7 days at 22 ° C along with a mixture of stabilizers containing 0.1-1% sorbitol and 0.1-1% glycine, added only in the process inactivation. Stabilizers in a specific concentration of 0.5% wt./about. glycine and 1% wt./about. sorbitol is added to live JE virus together with an inactivating agent, which is either formalin or BPL. Stabilizers are added to prevent degradation of the virus only during the incubation period for inactivation of 7 days, as mentioned above, due to the addition of an inactivating agent formalin or BPL. After inactivation, the mass of JE is diafiltered and concentrated by changing the buffer against phosphate-saline buffer repeatedly at least 5-6 times to remove formalin. If BPL is inactivated, diafiltration is not required. BPL is neutralized by incubating the mass at 37 ° C for 3 hours. The diafiltered mass of JE was sterilized by filtration using a 0.22 μm PVDF (polyvinylidene fluoride) membrane filter. The inactivated mass of Japanese encephalitis virus is then stored at 5 ° C ± 3 ° C for at least 24 months to 36 months. The stages of purification and inactivation of the mass of the virus of Japanese encephalitis strain Kolar according to one of the methods described in the framework of the present invention can be summarized below:

(a) purification of the virus collection using a 0.45 μm membrane to remove cell debris from the virus collection;

(b) column purification using combined size exclusion and chromatography with a Capto Core-700 matrix, wherein said matrix consists of a highly crosslinked agarose matrix with a functional active ligand octylamine group to collect the passed fraction containing purified virus;

(c) concentration and diafiltration of the passed fraction of stage (b) using 300-kDa cassettes with polyethersulfone membranes (PESU) to obtain purified concentrated viral mass;

(d) filtering the purified concentrated viral mass of step (c) through a 0.45 μm membrane of PVDF (polyvinylidene fluoride) to remove any impurities in the process;

(e) simultaneous stabilization and inactivation of the purified virus of stage (d) formalin in a ratio of from 1: 1500 vol./about. up to 1: 2500 rpm (i.e., 1 volume of formalin is added to 2500 volumes of live JE) together with stabilizers, where these stabilizers consist of glycine and sorbitol, for a period of 7 days at 22 ° C, or with beta-propiolactone in a ratio of 1: 1500 r ./about. up to 1: 2500 rpm (i.e., 1 volume of formalin is added to 2500 volumes of live JE) together with stabilizers, where said stabilizers consist of glycine and sorbitol, for a period of 7 days at 5 ° C ± 3 ° C;

(f) diafiltration and replacement of the inactivated purified virus mass buffer of step (e) using a 300-kDa cassette with a PESU membrane against phosphate buffered saline at least 5-6 times to remove only formalin if formalin inactivated;

(g) Filtration through a 0.22 μm membrane from PVDF (polyvinylidene fluoride) of the purified inactivated viral mass of step (f) to obtain a final and stabilized inactivated JEV mass at 5 ° C ± 3 ° C for at least 24 months to 36 months, free from any impurities, suitable for the preparation of a vaccine against Japanese encephalitis.

The tables below show the results of purification through a Celufine sulfate matrix for products with a batch size of 30 CF40, DB-100, DB-500 by repeated collection of the virus. The total viral mass obtained for one batch of 30 CF40 using Celufine sulfate in one month is listed below. Thus, the number of doses of JE vaccine that can be available with a 5 μg NMP per dose also increases significantly from about one hundred thousand doses in the case of 30 CF40 to at least 9 hundred thousand doses to 20 hundred thousand doses in the case of DB -100 to DB-500, respectively.

Table 3.1 The purified inactivated mass of JEV and the number of doses for 1 month using 30 CF40 by purification on a Capto Core-700 matrix. Formaldehyde inactivation and purification using captocore Titer Volume in liters Protein concentration, μg / ml Vh1 Virus collection 10 ^7.26 300 633.9 The fraction passed through the column - 326 442.8 Mass after dialysis through 0.22 μm / final mass - 8 168.75 Vh2 Virus collection 10 ^7.41 300 528.2 The fraction passed through the column - 331 379.2 Mass after dialysis through 0.22 μm / final mass - 7.2 175 Vh3 Virus collection 10 ^6.67 300 540.7 The fraction passed through the column - 408.3 Mass after dialysis through 0.22 μm / final mass - 6.3 181 Vh4 Virus collection 10 ^5.15 300 601 The fraction passed through the column - 312 399 Mass after dialysis through 0.22 μm / final mass - 5,0 150 Total protein (168.75 × 8) + (175 × 7.2) + (181 × 6.3) + (150 × 5.0) = 4500 mg Table 3.2 The purified inactivated mass of JEV and the number of doses for 1 month using DB-100 by purification on Capto Core-700. Formaldehyde inactivation and purification using captocore Titer Volume in liters Protein concentration, μg / ml Vh1 Virus collection 10 ^6.75 300 648.4 The fraction passed through the column - 327 481.6 Mass after dialysis through 0.22 μm / final mass - eighteen 391.5 Vh2 Virus collection 10 ^7.16 300 598.5 The fraction passed through the column - 320 448 Mass after dialysis through 0.22 μm / final mass - 13,2 193.5 Vh3 Virus collection 10 ^6-53 300 632.2 The fraction passed through the column - 311 476 Mass after dialysis through 0.22 μm / final mass - 5.9 900 Common Protein (391.5 × 18) + (193.5 × 13.2) + (900 × 5.9) = 675.0 mg Table 3.3 Purified inactivated mass of JEV and number of doses for 1 month using DB-500 by purification on Capto Core-700. Formaldehyde inactivation and purification using captocore Titer Volume in liters Protein concentration, μg / ml Vh1 Virus collection 10 ^6.82 500 598.6 The fraction passed through the column - 565 476.9 Mass after dialysis through 0.22 μm / final mass - 41 211 Vh2 Virus collection 10 ^6.26 500 540.8 The fraction passed through the column - 553 423.5 Mass after dialysis through 0.22 μm / final mass 31.5 160.1 Vh3 Virus collection 10 ^5-16 500 603.3 The fraction passed through the column - 527 485.8 Mass after dialysis through 0.22 μm / final mass - 17 103 Total protein (211 × 41) + (160.1 × 31.5) + (103 × 17) = 15445 mg

In addition, a comparison of the increase in the yield of the purified JEV mass using a Capto Core-700 column in the case of a batch size of 30 CF40, DB-100 and DB-500, respectively, is presented in the table below (table 3.4). You can see that the total mass of JEV in the case of 30 CF40 using capto core purification is 642857 mg, and in the case of DB-100 it is 964285 mg, which is equivalent to the production of 45 CF40 during the same required period of time, and 2206428 mg of inactivated mass of JEV which is equivalent to producing approximately 140 CF40 in the same required period of time. A summary of the comparison between the purification of Celufine sulfate and Capto Core 700 mass JEV is presented below:

Table 3.4 Comparison of the purified JEV mass obtained using a Celufine sulfate matrix and Capto Core 700 matrix. Batch device Total mass JE in mg
(Celufine sulfate column) Total mass JE in mg
(column Capto Core 700) Equivalent yield in cell factories Time for one commercial batch Increase in the number of times Output 30 CF40 450.64 642857 30 CF40 40 days About 10 times DB-100 output 722.56 964285 48 CF40 38 days About 9 times DB-500 output 2216.3 2206428 147 CF40 39 days About 8 times

Example 4: stability studies of the inactivated mass of JEV

The dialyzed inactivated mass of JEV was stored with or without suitable chemical stabilizer (s) at 5 ± 3 ° C. Antigen was selected at various intervals and examined by neutralizing plaque reduction (PRNT50). Antigen stability was determined using a viral dash of neutralizing mouse antiserum according to IP.2007; WHO TRS No.771. Virus neutralization in antiserum was determined by a 50% plaque reduction method. Virus neutralization titer was expressed as the inverse of the serum dilution, which showed a 50% reduction in plaques compared to the number of plaques in control wells without live viral antigen.

Table 4.1 The stability of the purified inactivated mass of JEV for 36 months Time Description pH Identification Abnormal toxicity Bacterial endotoxin Sterility Efficiency Particle-free white or substantially white clear liquid 6.50 to 7.50 Must meet performance test Must meet abnormal toxicity test Not more than 25 IU per single dose in humans Must satisfy sterility test NLT 50% PRNT induced by the study vaccine compared to the reference vaccine Zero day corresponds to 7.10 satisfies satisfies <25 IU on SHD satisfies Relative Efficiency 1.26 1 month corresponds to 7.07 satisfies NA NA NA Relative Efficiency 1.29 2 month corresponds to 7.03 satisfies NA NA NA Relative Efficiency28 3 month corresponds to 7.08 satisfies NA NA NA Relative Efficiency 1.24 6 month corresponds to 7.12 satisfies satisfies NA NA Relative efficiency 1.25 12 month corresponds to 7.12 satisfies NA NA NA Relative Efficiency 1.22 24 month corresponds to 7.09 satisfies satisfies <25 IU on SHD satisfies Relative Efficiency 1.24 36 month corresponds to 7.11 satisfies satisfies <25 IU on SHD satisfies Relative Efficiency 1.23

Claims (42)

1. Viral vaccine composition for the prevention of infections caused by the Indian strain of Japanese encephalitis virus JEV821564, comprising a vaccine antigen, wherein the vaccine antigen is an inactivated strain of Japanese encephalitis virus JEV821564 adapted for growth in Vero cells in a disposable bioreactor for collecting live weight of Japanese encephalitis virus. 2. The vaccine composition according to claim 1, where the specified vaccine antigen is grown in a disposable bioreactor in Vero cells to collect live weight of the Japanese encephalitis virus and the specified viral mass is collected repeatedly (multiple virus collection) from at least one rose up to five times or at least three times from one Vero cell culture and after a single infection with Japanese encephalitis virus strain JEV821564. 3. The vaccine composition according to claim 1, where the disposable bioreactor is supported with dissolved oxygen in the range of 50 ppm-70 ppm, a temperature range of 35.5 ° C to 37.5 ° C while maintaining a pH of 7, 2 to 7.6, with shaking from 260 rpm to 280 rpm, at an inlet flow rate of 80-150 ml / min and an outlet flow rate of 84-156 ml / min. 4. The vaccine composition according to claim 2, where the resulting multiple viral collections are further purified and inactivated, through the steps of: (a) purification of the virus collection using a 0.45 μm membrane to remove cell debris from the virus collection; (b) concentrating the collection volume at least 4 times using a 300-kDa membrane cassette to obtain concentrated retentate; (c) purification on a column of the concentrated collected retentate of step (b) using a Celufine sulfate matrix, wherein said matrix consists of a matrix with a cellulose support in the presence of an activated group sulfate ester to collect the eluate containing the desired virus, a high amount of cell salts proteins and nucleic acid material; (d) diafiltration and buffer replacement using a 300-kDa cassette against the phosphate-salt buffer of the eluate of step (c) for at least 5-6 times to collect retentate to remove unwanted high amounts of salts of cellular proteins and nucleic acid material from virus of interest for collecting purified concentrated virus mass; (e) filtering the purified concentrated viral mass of step (d) through a 0.45 μm membrane to eliminate any impurities in the process; (f) the simultaneous stabilization and inactivation of the purified virus of stage (e) formalin in a ratio of from 1500: 1 vol./about. up to 2500: 1 v / v together with stabilizers, where these stabilizers consist of glycine and sorbitol, for a period of 7 days at 22 ° C, or with beta-propiolactone in a ratio of from 1500: 1 vol./about. up to 2500: 1 v / v together with stabilizers, where these stabilizers are composed of glycine and sorbitol, for a period of 7 days at 5 ° C ± 3 ° C; (g) diafiltration and replacement of the inactivated purified virus mass buffer of step (f) using a 300-kDa cassette with a membrane against phosphate-saline buffer at least 5-6 times to remove only formalin in case of formalin inactivation; (h) filtering through a 0.22 μm membrane of the purified inactivated viral mass of step (g) to obtain a final and stabilized JEV mass at 5 ° C ± 3 ° C for at least 24 months to 36 months, free of any or impurities suitable for the preparation of a vaccine against Japanese encephalitis. 5. The vaccine composition according to claim 2, where the resulting multiple viral collections are further purified and inactivated by the steps of: (a) purification of the virus collection using a 0.45 μm membrane to remove cell debris from the virus collection; (b) column purification using combined size exclusion and affinity chromatography, wherein said matrix consists of a highly crosslinked agarose matrix with a functional active ligand octylamine group to collect the passed fraction containing purified virus; (c) concentration and diafiltration of the passed fraction of stage (b) using 300-kDa membrane cassettes to obtain purified concentrated viral mass; (d) filtering the purified concentrated viral mass of step (c) through a 0.45 μm membrane to eliminate any impurities in the process; (e) simultaneous stabilization and inactivation of the purified virus of stage (d) formalin in a ratio of from 1500: 1 v / v. up to 2500: 1 v / v together with stabilizers, where these stabilizers consist of glycine and sorbitol, for a period of 7 days at 22 ° C, or with beta-propiolactone in a ratio of from 1500: 1 vol./about. up to 2500: 1 v / v together with stabilizers, where these stabilizers are composed of glycine and sorbitol, for a period of 7 days at 5 ° C ± 3 ° C; (f) diafiltration and replacement of the inactivated purified virus mass buffer of step (e) using a 300-kDa cassette with a membrane against phosphate-saline buffer at least 5-6 times to remove only formalin in case of formalin inactivation; (g) filtering through a 0.22 μm membrane of the purified inactivated viral mass of step (f) to obtain a final and stabilized inactivated JEV mass at 5 ° C ± 3 ° C for at least 24 months to 36 months, free of any or any impurities suitable for the preparation of a vaccine against Japanese encephalitis. 6. A method of producing a viral vaccine composition by adapting a strain of Japanese encephalitis virus JEV821564 to Vero cells capable of industrial production in a disposable bioreactor to collect live weight of Japanese encephalitis virus, said viral mass being collected multiple times (multiple virus collection) from at least more than one up to five times or at least three times from one Vero cell culture and upon a single infection with the Japanese encephalitis virus strain JEV821564. 7. The method according to claim 6, where multiple mass collection of Japanese encephalitis virus is scaled from laboratory scale batches in a disposable bioreactor with a size equivalent to 4 cell factories to produce batch sizes in a disposable bioreactor with a size equivalent to about 200 cell factories. 8. The method according to claim 6 and 7, where the disposable bioreactor is supported with dissolved oxygen in the range of 50 ppm-70 ppm, a temperature range of 35.5 ° C to 37.5 ° C while maintaining a pH of 7 , 2 to 7.6, with shaking from 260 rpm to 280 rpm, at an inlet flow rate of 80-150 ml / min and an outlet flow rate of 84-156 ml / min. 9. The method according to claim 7, where multiple mass collection of Japanese encephalitis virus is obtained at least three times in a disposable bioreactor with a size equivalent to approximately 200 cell factories for a period of at least 35-40 days. 10. The method of simultaneous cleaning and inactivation of the collection of mass of the virus of Japanese encephalitis according to claim 7, comprising the steps of: (a) purification of the virus collection using a 0.45 μm membrane to remove cell debris from the virus collection; (b) concentrating the collection volume at least 4 times using a 300-kDa membrane cassette to obtain concentrated retentate; (c) purification on a column of the concentrated collected retentate of step (b) using a Celufine sulfate matrix, wherein said matrix consists of a matrix with a cellulose support in the presence of an activated group sulfate ester to collect the eluate containing the desired virus, a high amount of cell salts proteins and nucleic acid material; (d) diafiltration and buffer replacement using a 300-kDa cassette against the phosphate-salt buffer of the eluate of stage (c) for at least 5-6 times to collect retentate to remove unwanted high amounts of salts of cellular proteins and nucleic acid material from virus of interest for collecting purified concentrated virus mass; (e) filtering the purified concentrated viral mass of step (d) through a 0.45 μm membrane to eliminate any impurities in the process; (f) the simultaneous stabilization and inactivation of the purified virus of stage (e) formalin in a ratio of from 1500: 1 vol./about. up to 2500: 1 v / v together with stabilizers, where these stabilizers consist of glycine and sorbitol, for a period of 7 days at 22 ° C, or with beta-propiolactone in a ratio of from 1500: 1 vol./about. up to 2500: 1 v / v together with stabilizers, where these stabilizers are composed of glycine and sorbitol, for a period of 7 days at 5 ° C ± 3 ° C; (g) diafiltration and replacement of the inactivated purified virus mass buffer of step (f) using a 300-kDa cassette with a membrane against phosphate-saline buffer at least 5-6 times to remove only formalin in case of formalin inactivation; (h) filtering through a 0.22 μm membrane of the purified inactivated viral mass of step (g) to obtain a final and stabilized JEV mass at 5 ° C ± 3 ° C for at least 24 months to 36 months, free of any or impurities suitable for the preparation of a vaccine against Japanese encephalitis. 11. The method of simultaneous purification and inactivation of the collection of virus masses of Japanese encephlitis virus according to claim 7, comprising the steps of: (a) purification of the virus collection using a 0.45 μm membrane to remove cell debris from the virus collection; (b) column purification using combined size exclusion and affinity chromatography, wherein said matrix consists of a highly crosslinked agarose matrix with a functional active ligand octylamine group to collect the passed fraction containing purified virus; (c) concentration and diafiltration of the passed fraction of stage (b) using 300-kDa membrane cassettes to obtain purified concentrated viral mass; (d) filtering the purified concentrated viral mass of step (c) through a 0.45 μm membrane to eliminate any impurities in the process; (e) simultaneous stabilization and inactivation of the purified virus of stage (d) formalin in a ratio of from 1500: 1 v / v. up to 2500: 1 v / v together with stabilizers, where these stabilizers consist of glycine and sorbitol, for a period of 7 days at 22 ° C or with beta-propiolactone in a ratio of 1500: 1 vol./about. up to 2500: 1 v / v together, the stabilizers, where these stabilizers consist of glycine and sorbitol, for a period of 7 days at 5 ° C ± 3 ° C; (f) diafiltration and replacement of the inactivated purified virus mass buffer of step (e) using a 300-kDa cassette with a membrane against phosphate-saline buffer at least 5-6 times to remove only formalin in case of formalin inactivation; (g) filtering through a 0.22 μm membrane of the purified inactivated viral mass of step (f) to obtain a final and stabilized inactivated JEV mass at 5 ° C ± 3 ° C for at least 24 months to 36 months, free of any or any impurities suitable for the preparation of a vaccine against Japanese encephalitis. 12. A method for increasing the commercial production of a viral vaccine composition obtained from the purified inactivated mass of Japanese encephalitis virus (JEV) strain JEV821564 in a single batch in a single-use bioreactor up to at least 8 times to 10 times using combined exclusion and affinity chromatography, where the specified matrix consists of a highly cross-linked agarose matrix with a functional active ligand octylamine group, for a period of 35-40 days, compared with purified inacti th e weighing JEV, obtained with a commercial product in one batch size JEV in 30 cell factories during the same period of time of 35-40 days.